Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco.

S.Saschenbrecker,A.Bracher,K.V.Rao,B.V.Rao,F.U.Hartl,M.Hayer-Hartl.

ABSTRACT

After folding, many proteins must assemble into oligomeric complexes to become
biologically active. Here we describe the role of RbcX as an assembly chaperone
of ribulose-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme responsible
for the fixation of atmospheric carbon dioxide. In cyanobacteria and plants,
Rubisco is an approximately 520 kDa complex composed of eight large subunits
(RbcL) and eight small subunits (RbcS). We found that cyanobacterial RbcX
functions downstream of chaperonin-mediated RbcL folding in promoting the
formation of RbcL(8) core complexes. Structural analysis revealed that the 15
kDa RbcX forms a homodimer with two cooperating RbcL-binding regions. A central
cleft specifically binds the exposed C-terminal peptide of RbcL subunits,
enabling a peripheral surface of RbcX to mediate RbcL(8) assembly. Due to the
dynamic nature of these interactions, RbcX is readily displaced from RbcL(8)
complexes by RbcS, producing the active enzyme. The strategies employed by RbcX
in achieving substrate specificity and efficient product release may be
generally relevant in assisted assembly reactions.

Selected figure(s)

Figure 4.
Figure 4. Binding of C-Terminal RbcL Peptide to RbcX (A)
A cellulose membrane containing an array of overlapping
dodecamer peptides covering the sequence of Syn7002-RbcL was
probed with the RbcX proteins indicated. Peptide-bound RbcX was
visualized by chemiluminescent immunodetection with anti-RbcX
antibody. (B) Alignment using MultAlin (Corpet, 1988) of
C-terminal amino acid sequences of RbcL from the cyanobacterial
and higher plant species indicated (Swiss-Prot accession numbers
in brackets). High consensus level (≥ 90%) is depicted in red
and low consensus level (≥ 50%) in blue. (C) Structure
of the complex of peptide EIKFEFD bound to RbcX dimer. The
peptide is shown in stick representation; RbcX is represented as
a molecular surface with protomers colored white and blue,
respectively. N and C termini of the peptide are indicated.
(D) Magnification of boxed area in (C) presenting a view of the
refined peptide bound to RbcX. Molecular interactions between
peptide and RbcX are highlighted. Dashed lines represent
hydrogen bonds. Residues of the RbcX monomers participating in
peptide binding are displayed in stick representation below the
transparent surface of the molecule and are numbered in white
and yellow, respectively. The important hydrophobic residues in
the bound peptide are also labeled.

Figure 7.
Figure 7. Working Model of RbcX Function in Cyanobacterial
Rubisco Assembly RbcX functions to increase the efficiency
of Rubisco assembly by acting on folded RbcL subunits subsequent
to their GroEL/GroES-mediated folding. Recognition of RbcX
requires the exposed C-terminal RbcL peptide (see Discussion for
details and Figure S11). Note that assembly of RbcL[8]S[8] may
also occur independently of RbcX for some Rubisco homologs,
presumably involving similar assembly intermediates.

A.M.Hirtreiter,G.Calloni,F.Forner,B.Scheibe,M.Puype,J.Vandekerckhove,M.Mann,F.U.Hartl,andM.Hayer-Hartl
(2009).Differential substrate specificity of group I and group II chaperonins in the archaeon Methanosarcina mazei.

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